Method and compositions for enhancing aminolevulinic acid dehydratase assay
The present invention provides a method of improving the sensitivity and accuracy of a lead assay. The method enhances the recovery of lead during isolation of the lead from interfering compounds by maintaining the lead in a sample solution and making the recovered lead available for detection by the assay. An enhancing reagent complexes with the lead isolated in the sample solution. The enhancer includes a chelator having a lead equilibrium binding constant in the range of about 4 log K to about 13 log K. A kit for performing such a lead assay is also provided.
Latest Abbott Laboratories Patents:
- Methods for aiding in diagnosing and evaluating a mild traumatic brain injury in a human subject using cardiac troponin I and early biomarkers
- Nucleic acid amplification and detection devices, systems and methods
- Apparatus for removing liquid contents of a container
- CONTAINER WITH A LID-COLLAR ASSEMBLY
- NUTRITIONAL COMPOSITIONS COMPRISING HUMAN MILK OLIGOSACCHARIDES AND NUCLEOTIDES AND USES THEREOF FOR TREATING AND/OR PREVENTING ENTERIC VIRAL INFECTION
Claims
1. A lead assay comprising the steps of:
- (a) providing an aqueous solution suspected of containing lead;
- (b) isolating said lead from said solution in such a manner that said lead remains in solution:
- (c) introducing to said solution of step (b) an enhancing reagent that combines with said lead and prevents said lead from precipitating from said solution; followed by
- (d) introducing to said solution an enzyme the activity of which is inhibited in the presence of lead and a substrate for said enzyme: and
- (e) measuring the amount of lead as a function of said activity of said enzyme.
2. The lead assay of claim 1 wherein the reagent which combines with lead in step (b) comprises a chelator having a lead equilibrium binding constant in the range of about 4 log K to aout 13 log K.
3. The lead assay of claim 2 wherein the lead equilibrium binding constant is in the range of about 6 log K to about 9 log K.
4. The lead assay of claim 2 wherein the chelator is selected from the group consisting of N-benzyliminodiacetic acid, ethylenebis(oxyethylenenitrilo) tetraacetic acid, ethylenediaminetetraacetic acid, L-histidine monohydrochloride monohydrate, N-(2-hydroxyethyl)-iminodiacetic acid, iminodiacetic acid, DL-penicillamine, methyliminodiacetic acid, nitrilotriacetic acid, sodium citrate, and d-hydroxyquinoline-5-sulfonic acid hydrate.
5. A lead assay comprising the steps of:
- (a) providing an aqueous solution suspected of containing lead, said solution having been separated from compounds that are affected by the presence of lead, said solution further having been neutralized;
- (b) introducing into said solution a lead chelator having a lead equilibrium binding constant in the range of about 4 log K to about 13 log K; followed by
- (c) introducing into said solution (i) an enzyme the activity of which is inhibited by lead and (ii) a substrate which reacts with the enzyme;
- (d) incubating the solution of step (c):
- (e) stopping the incubation step after a predetermined interval; and
- (f) measuring the amount of lead as a function of enzyme activity.
6. The lead assay of claim 5 wherein the isolating step includes releasing lead from a sample of whole blood.
7. The lead assay of claim 5 wherein the method further includes neutralizing the sample solution before the enzyme incubating step.
8. The lead assay of claim 5 wherein the chelator has a lead equilibrium binding constant in the range of about 6 log K to about 9 log K.
9. The method of claim 5 wherein the chelator is selected from the group consisting of N-benzyliminodiacetic acid, ethylenebis(oxyethylenenitrilo) tetraacetic acid, ethylenediaminetetraacetic acid, L-histidine monohydrochloride monohydrate, N-(2-hydroxyethyl)-iminodiacetic acid, iminodiacetic acid, DL-penicillamine, methyliminodiacetic acid, nitrilotriacetic acid, sodium citrate, and d-hydroxyquinoline-5-sulfonic acid hydrate.
10. The method of claim 5 wherein the chelator is selected from the group consisting of 8-hydroxy-5-(2'-hydroxyphenylazo)quinoline, 8-hydroxy-5-(phenylazo)quinoline, N-(2-carboxyphenyl)iminodiacetic acid, N-(acetonyl)iminodiacetic acid, N-(dithiocarboxy)aminoacetic acid, N,N-bis(2'-hydroxyethyl)glycine, and glycine.
11. The method of claim 5 wherein the chelator is selected from the group consisting of dihydroxyphenyl acetic acid, N-(2'-carboxyethyl) iminodiacetic acid, dihydroxybenzoic acid, 3,4,dihydroxybenzene sulfonic acid, melonic acid, 1-hydroxy-1-(3'-pyridyl) methane sulfonic acid, and 4-aminopyridine-2,6-dicarboxylic acid.
12. The lead assay of claim 5 wherein the aqueous solution in step (b) is acidified and the lead chelator of step (c) is present in a neutralizing buffer such that performing step (c) results in bringing the acidified solution of step (b) to neutral pH.
13. The lead assay of claim 5 wherein the enzyme is aminolevulinic acid dehydratase.
14. The lead assay of claim 13 wherein the enzyme is activated with a reducing agent selected from the group consisting of dithiothreitol, glutathione, mercaptoethanol and cysteine.
15. The lead assay of claim 5 wherein the enzyme incubating step includes first incubating the sample solution in the presence of aminolevulinic acid dehydratase and subsequently incubating the sample solution in the presence of the substrate.
16. The lead assay of claim 15 wherein the substrate includes aminolevulinic acid.
17. The lead assay of claim 5 wherein the stopping step includes adding a stop reagent.
18. The lead assay of claim 17 wherein the stop reagent includes HgCl.sub.2.
19. The lead assay of claim 5 wherein step (d) includes incubating the sample solution in the presence of a coloring reagent.
20. The lead assay of claim 19 wherein the coloring reagent is selected from the group consisting essentially of dimethylaminobenzaldehyde and dimethylaminocinnamaldehyde.
21. An aqueous lead assay reagent solution consisting essentially of neutralizing buffer and a lead chelator having a lead binding constant in the range of about 4 log K to about 13 log K wherein the concentration of the chelator in the solution is in the range of 0.5 mM to 500 mM.
22. The reagent solution of claim 21 wherein the chelator has a lead equilibrium binding constant in the range of about 6 log K to about 9 log K.
23. The reagent solution of claim 21 wherein the chelator is selected from the group consisting of N-benzyiminodiacetic acid, ethylenebis(oxyethylenenitrilo) tetraacetic acid, ethylenediaminetetraacetic acid, L-histidine monohydrochloride monohydrate, N-(2-hydroxyethyl)-iminodiacetic acid, iminodiacetic acid, DL-penicillamine, methyliminodiacetic acid, nitrilotriacetic acid, sodium citrate, and d-hydroxyquinoline-5-sulfonic acid hydrate.
24. The reagent solution of claim 23 wherein the chelator is selected from the group consisting of 8-hydroxy-5-(2'-hydroxyphenylazo)quinoline, 8-hydroxy-5-(phenylazo)quinoline, N-(2-carboxyphenyl)iminodiacetic acid, N-(acetonyl) iminodiacetic acid, N-(dithiocarboxy) aminoacetic acid, N,N-bis(2'-hydroxyethyl)glycine, and glycine.
25. The reagent solution of claim 21 wherein the chelator is selected from the group consisting of dihydroxyphenyl acetic acid, N-(2'-carboxyethyl) iminodiacetic acid, dihydroxybenzoic acid, 3,4,dihydroxybenzene sulfonic acid, melonic acid, 1-hydroxy-1-(3'-pyridyl)methane sulfonic acid, and 4-aminopyridine-2,6-dicarboxylic acid.
26. A lead assay reagent kit comprising:
- a container having a reagent solution consisting essentially of an aqueous neutralizing buffer and present therein a reagent which is capable of forming a compound or complex with lead such that adding the solution to an acidified aqueous sample containing lead will neutralize the aqueous sample while preventing precipitation of lead therefrom;
- a container comprising an enzyme which is inhibited in the presence of lead; and
- a container comprising a substrate which reacts with said enzyme.
27. The lead assay reagent kit of claim 26 wherein the kit further includes:
- a container for a stop reagent for stopping the reaction between the substrate and the enzyme;
- a container for a coloring reagent for forming a chromophore upon reaction with a product of the substrate and enzyme reaction; and;
- a reducing reagent included in either the substrate or enzyme containiner in an amount effective to increase the activity of the enzyme reacting with the substrate.
28. The lead assay reagent kit of claim 27 wherein the reducing agent comprises dithiothreitol, the enzyme comprises aminolevulinic acid dehydratase and the substrate comprises aminolevulinic acid and the product of the substrate and enzyme comprises porphobilinogen.
| 4042329 | August 16, 1977 | Hochstrasser |
| 4203971 | May 20, 1980 | Buchanan |
| 4912035 | March 27, 1990 | Belly et al. |
| 5112749 | May 12, 1992 | Brey, III et al. |
| 5151412 | September 29, 1992 | Brown |
| 5354652 | October 11, 1994 | Silbergeld |
| 5368707 | November 29, 1994 | Henkens et al. |
| WO93/01310 | January 1993 | WOX |
- Odgen et al, Methods in Enzymology, vol. 152, p. 65, 1987. Joffe et al, Biological Trace Element Research, vol. 28, No. 3, pp. 223-231, Mar. 1991. Silbergeld, Chemical Abstracts, vol. 118, p. 345, Ref. #185700z, 1993 (PCT Int. App. WO 9301310, 21 Jan. 1993). Cavallerr et al, Chemical Abstracts, vol. 88, pp. 385-386, Ref. #1764324, 1978 (Br. J. Ind. Med. 1978, 351), 21-6. Despaux-Rogers et al, Chemical Abstracts, vol. 105, p. 298, Ref. #37935w (Int. Arch. Occup. Enviorn. Health 1986, 57(4) 303-13). A. Berlin, et al., European Standarized Method for the Determination of .delta.-Aminolevulinic Acid Dehydratase Activity in Blood, Z. Klin. Chem. Klin. Biochem. S. 389-390 (1974). P. N. B. Gibbs, et al., Purification and properties of 5-aminolaevulinate dehydratase from human erythrocytes, Biochem. J., 230, 25-34 (1985). S. Sassa, Delta-Aminolevulinic Acid Dehydratase Assay, Enzyme 28, 133-145 (1982). P. M. Anderson, et al., Purification and Properties of .delta.-Aminolevulinate Dehydrase from Human Erythrocytes, The Journal of Biological Chemistry, vol. 254, No. 15, Issue of Aug. 10, 6924-6930 (1979). M. T. Volosin, et al., Use of the Carbon Rod Atomizer for Analysis of Lead in Blood: Three Methods Compared, Clinical Chemistry, vol. 21, No. 13, 1986-1987 (1975). J. O. Pierce, et al., Lead, Chromium, and Molybdenum by Atomic Absorption, Arch Environ Health, vol. 13, 208-212 (Aug. 1966).
Type: Grant
Filed: Jul 26, 1995
Date of Patent: Oct 13, 1998
Assignee: Abbott Laboratories (Abbott Park, IL)
Inventors: Martin Wong (Grayslake, IL), David M. Finley (Spring Grove, IL)
Primary Examiner: Howard E. Schain
Assistant Examiner: Abdel A. Mohamed
Attorney: David L. Weinstein
Application Number: 8/507,168
International Classification: C12Q 134; G01N 3300;
